Fc receptor-mediated antibody functions may play a role in preventing HIV infection. One such function, antibody-dependent cell-mediated phagocytosis (ADCP), occurs when antibody binds a pathogen or a target cell expressing foreign antigens, allowing crosslinking of Fc receptors on phagocytes and the subsequent internalization of the pathogen or target cell. ADCP likely participates in the clearance of many viruses. However, a role for ADCP in preventing HIV-1 infection has not been established. In this R03 project, we will begin to assess the potential impact of ADCP on the in vitro clearance of HIV-1. Recent ADCP assays rely on 1 M beads coated with monomeric Env glycoprotein. However, the biological targets of ADCP-virions and infected cells-are much smaller and much larger, respectively, than 1 M beads, and pertinent antigens are in the trimeric Env. Thus, one objective of our proposal is to develop biologically relevant assays to measure ADCP. We will test the hypothesis that authentic HIV-1 virions, infected cells, and cells decorated with HIV-1 can be used to reproducibly measure ADCP antibody activity. Our preliminary data indicate that there is inefficient ADCP of antibody-opsonized virions, whereas ADCP of infected cells or of T-cells decorated with virions occurs efficiently. These and published observations suggest that Env density and the capacity to form aggregates are critical determinants of virion ADCP. Thus, we will also test the hypothesis that the small number of Env spikes on HIV-1 virions restricts virus aggregation by antibody and subsequent crosslinking of Fc receptors on phagocytes, thereby limiting ADCP potency. To test these hypotheses, we will accomplish the following: Specific Aim 1: develop reproducible, biologically relevant assays to measure ADCP of HIV-1 virions, infected cells, and cells decorated with HIV-1 virions; and Specific Aim 2: define the conditions that limit the potency of ADCP against HIV-1 virions. To our knowledge, the proposed research would be the first to systematically investigate ADCP of HIV-1 virions and cells bearing authentic Env glycoproteins in a biologically relevant manner. Since ADCP may be a key protective antibody function, developing assays that reliably ascertain its role as a correlate of protection after vaccination will have a great impact on the HIV vaccine field. Moreover, it is likely that critical insights into how antibodies clear virus and infected cells wil emerge from these studies.